Journal of Toxicology and Environmental Health
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Cd‐metallothionein nephrotoxicity in inbred strains of mice L.E. Sendelbach , W.C. Kershaw , F. Cuppage & C.D. Klaassen To cite this article: L.E. Sendelbach , W.C. Kershaw , F. Cuppage & C.D. Klaassen (1992) Cd‐metallothionein nephrotoxicity in inbred strains of mice, Journal of Toxicology and Environmental Health, 35:2, 115-126, DOI: 10.1080/15287399209531600 To link to this article: http://dx.doi.org/10.1080/15287399209531600
Published online: 19 Oct 2009.
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Date: 06 November 2015, At: 03:52
Cd-METALLOTHIONEIN NEPHROTOXICITY IN INBRED STRAINS OF MICE
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L. E. Sendelbach, W. C. Kershaw, F. Cuppage, C. D. Klaassen Environmental Health and Occupational Medicine Center, Department of Pharmacology, Toxicology, and Therapeutics, Department of Pathology and Oncology, University of Kansas Medical Center, Kansas City, Kansas
Genetic differences in the acute hepatic and testicular toxicity of Cd occur among different strains of mice. However, it is not known whether genetic variation to the renal damage caused by Cd-metallothionein (CdMT) exists. Therefore, male mice of the C3H/HeJ,C57/B/10, CBA/CA, and DBA/2] strains, previously shown to differ in hepatic and testicular injury due to Cd, were treated with CdMT at dosages of 0.2, 0.4, 0.8, and 1.6 mg/kg (sc). For all strains of mice, tissue accumulation of Cd occurred predominantly in kidney, which had two to three times as much Cd as liver, while testes had no measurable amounts of Cd. Hepatic and renal metallothionein (MT) concentrations were increased with increasing dosage of CdMT, and no differences between strains were demonstrated. Urinary glucose was increased significantly at the three highest dosages of CdMT, with no differences between strains. At each dose level, light microscopic manifestations of CdMT nephropathy did not differ between strains. In summary, all CdMT-treated strains of mice responded similarly with respect to all measured renal parameters (accumulation of Cd and MT and nephrotoxicity). Unlike the strain differences in hepatic and testicular injury from Cd in these strains of mice, CdMT nephrotoxicity shows no such genetic variation.
INTRODUCTION Metallothionein (MT) is a low-molecular-weight protein capable of binding divalent metals. This metal-binding capacity protects experimental animals against Cd toxicity. Cd binds to hepatic MT, forming a cadmium-metallothionein (CdMT) complex within liver that renders the ion nontoxic (Goering and Klaassen, 1984). Some of the CdMT complex is released from hepatic tissue and eventually deposits within the proximal tubule cells of the kidney (Dudley et al., 1985). Relocation of CdMT from This work was supported by the U.S. Public Health Service grant ES-01142. Presented in part at the 27th Annual Meeting of the Society of Toxicology, February 1988, Dallas, Tex. L. E. Sendelbach was supported by U.S. Public Health Service training grant ES-07079, and has a current address of 57 Union Street, Worcester, MA 01608. W. C. Kershaw was supported by U.S. Public Health Service grant ES-07079, and has a current address of Miami Valley Laboratories, Procter & Gamble Company, Cincinnati, OH 45239. Requests for reprints should be sent to C. D. Klaassen, Ph.D., Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, 39th and Rainbow Blvd., Kansas City, KS 66160-7417. 115 Journal of Toxicology and Environmental Health, 35:115-126, 1992 Copyright © 1992 by Hemisphere Publishing Corporation
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the liver to the kidney might be responsible for the chronic toxicity of Cd seen in humans and experimental animals. In contrast to the nephrotoxic potential of CdMT, the target tissues of inorganic Cd (e.g., CdCl2) are testes and liver. Genetic differences of Cd toxicity in testes and liver have been demonstrated in certain strains of mice. Gunn et al. (1965,1968) have shown the testes of rodents to be particularly sensitive to inorganic Cd. In addition, some strains of mice show particular sensitivities to Cd-induced testicular damage. The AJ, BALB/CJ, C57BL/6J, and C3H/HeJ strains are resistant to testicular damage, whereas the 129/J, AKR/J, CBA/J, and C57BR/cdJ are sensitive (Gunn et al., 1965, 1968; Hata et al. 1978; Chellman et al., 1984, 1985; Nolan and Shaikh, 1986). The differences in sensitivities of murine strains to Cd-induced testicular damage lead Taylor to propose that this response is genetically controlled by a single autosomal recessive gene designated cdm (Taylor et al., 1973). Strain variations in the response of hepatic tissue of inorganic Cd are evident, as well. The C3H strain is particularly sensitive to Cd-induced hepatotoxicity (Quaife et al., 1984), whereas the DBA and BALB/c strains are more resistant to the hepatotoxic action of Cd (Tsunoo et al., 1979). However, those strains of mice that are resistant to hepatic damage are not necessarily resistant to testicular damage caused by Cd. For example, the C3H strain is resistant to Cd-induced testicular damage but hypersensitive to hepatic damage. Although genetic variation to the hepatic and testicular damage of Cd is evident in certain strains of mice, there is no known genetic variation to the renal damage caused by CdMT. Because CdMT is the organic form of Cd suspected to be responsible for chronic nephrotoxicity (Dudley et al., 1985), a genetic analysis of murine strain variation might be beneficial in understanding the mechanism of chronic Cd disease. Therefore, mice of the C3H/HeJ, C57BL/10, CBA/CA, and DBA/ 2J strains, previously shown to differ in hepatic and testicular injury due to Cd, were treated with CdMT to test for strain variation to this form of Cd.
METHODS Animals Male C3H/HeJ, C57BL/10, CBA/CA, and DBA/2J mice were received from Jackson Laboratories (Bar Harbor, Me.). All animals were 6 wk of age at receipt, and housed for a 2-week acclimation period. Animals were group housed in plastic shoebox cages on hardwood chip bedding. Fluorescent lighting was maintained on a 12-h on/off cycle. Food, in pellet form (Purina Rodent Laboratory Chow 5001, Ralston Purina Co., St. Louis,
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Mo.), and water were provided ad libitum. The temperature of the animal room was maintained at 23 ± 1°C. Mice were injected with CdCI2 (25 /*mol/kg, sc, n = 6), 109CdMT (0.2, 0.4, 0.8, and 1.6 mg Cd/kg, sc, n = 6), or isotonic saline as vehicle controls (n = 4). Serum was collected for evaluation of transaminase activity 24 h following CdCI2 or CdMT administration. Samples were stored at 4°C and assayed within 24 h of collection. Immediately following serum collection, testes and livers were then removed and weighed. Kidneys were then removed and stored in 10% neutral buffered formalin. Kidneys were routinely processed, stained with hematoxylin and eosin, and evaluated microscopically. Chemicals Cadmium chloride was obtained from Fisher Scientific Co. (St. Louis, Mo.). Carrier-free 109Cd was obtained from New England Nuclear (Boston, Mass.). The reagents used for the determination of glucose and creatinine in urine as well as the activities of alanine (ALT) and aspartate aminotransferases (AST) in serum were supplied as kits (Sigma Chemical Co., St. Louis, Mo.). All other chemicals were of reagent grade. Preparation of Cd-Metallothionein Male rats received 3 mg Cd/kg, sc, plus 1.6 /xCi 109Cd/kg once a day, 3 times per week for 3 wk. Rats were decapitated and the livers were excised and homogenized in a Potter-Elvehjem glass homogenizer, using a Teflon pestle, in a 1 :1 volume of Tris-acetate buffer (pH 7.4, 4°C). The homogenate was then centrifuged at 10,000 x g for 10 min (4°C) in a Sorvall RC2-B centrifuge (Sorvall Instruments, Norwalk, Conn.). The supernatant was heat treated at 80°C for 5 min followed by centrifugation at 15,000 x g for 20 min. This supernatant was then applied to a Sephadex G-75 column (2.6 x 60 cm) and eluted with 10 mM "iris-acetate buffer (pH 7.4, 4°C). The MT peak obtained from the column, with a relative elution volume (Ve/Vo) of approximately 2, was collected, applied to a DEAE Sephadex A-25 column (2.6 x 35 cm), and eluted with a linear gradient of 10-240 mM Tris-acetate buffer (pH 7.4, 4°C). Fractions from the second peak (MT-II) were pooled, desalted through G-25 columns, and lyophilized. Tissue Cd Concentration Portions of the testes, kidney, and liver were assayed for 109Cd radioactivity by means of a Packard model 5130 Autogamma scintillation counter (Packard Instrument Co., Downers Grove, III.), and tissue Cd contents were calculated using the specific activity of the 109Cd-MT dosing solutions.
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Metallothionein Analysis Mice were necropsied 24 h following CdMT administration and the testes, kidney, and liver were excised for determination of MT content. Metallothionein determinations were performed by the Cd-hemoglobin assay of Onasaka et al. (1978) as described by Eaton and Toal (1982).
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Urinalysis Mice were administered Cd-MT, and hydrated 22 h later at a volume of 50 ml/kg. Urine was collected over a 2-h period according to the method of Plaa and Larson (1965) without filter paper. Biochemical Assays Serum activities of ALT and AST were determined according to the method of Bergmeyer et al. (1978). Urinary glucose (Sigma technical bulletin 15-UV, 1983) and creatinine (Sigma procedure 555) were determined using commercially available kits (Sigma Chemical Co.). Macro and Microscopic Assessment of Testicular and Renal Tissues Testes were evaluated for surface hemorrhage at gross necropsy. Grade scores of 1, 2, 3, 4, and 5 corresponded to 0-5, 5-25, 25-50, 50-75, and >75%, respectively, of the testicular surface being hemorrhagic. Kidneys were fixed in 10% neutral buffered formalin, routinely processed, sectioned at 6 /*m, and stained with hematoxylin and eosin for microscopic assessment. Lesions were scored, using 1 to indicate normal, 2 for focal renal tubular necrosis (75%) of the proximal tubule with all components of the tubules necrotic with multiple casts in distal segments of the nephrons. Statistics Data are presented as mean ± (SE). The data were initially compared using a one-way analysis of variance. When significant differences were found, intrastrain comparisons were made using the post hoc procedure of Dunnett's multiple comparison test; interstrain comparisons were performed by the post hoc procedure of Duncan's multiple range test. Differences were considered significant at p < .05. RESULTS Strain Comparison of CdCI2 Toxicity Hepatotoxicity Figure 1 illustrates the hepatic toxicity of CdCI2 in C3H, CBA, DBA, and C57 strains of mice, as measured by the serum activities of ALT and AST. CdCI2, at 25 jumol/kg, increased the activities of
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Saline
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CdCl,
FIGURE 1. Hepatic injury produced by CdCI2. Mice were treated with CdCI2 (25 ftmol/kg, sc), and sera were collected 24 h later for measurement of aspartate aminotransferase (AST, upper panel), and alanine aminotransferase (AST, lower panel) activities. Values represent means (SE) of six mice from each strain. Bars with differing letters differ significantly, p < .05, according to Duncan's multiple range test.
both serum enzymes in all four strains of mice 24 h following administration. CdCI2 produced a greater increase in ALT and AST in the C3H strain than in the other strains. Thus the C3H strain is hypersusceptible to the hepatic effects of Cd, as had been demonstrated previously (Quaife et al., 1984). Testicular Toxicity Strain variation to testicular toxicity of CdCI2 is depicted in Figure 2. The toxicity of CdCI2 to testicular tissues was determined quantitatively by an increase in testicular weights and qualitatively by severity of testicular surface hemorrhage. Both measurements of toxicity were increased in the CBA and DBA mice, strains known to be sensitive to the testicular effects of Cd (Taylor et al., 1973; Nolan et al., 1986). The C3H and C57 murine strains were resistant to the testicular
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300
Saline
CdCU
FIGURE 2. Testicular damage induced by CdCI2. Mice were treated with CdCI2 (25 ^mol/kg, sc), and testes were collected 24 h later. Testes were evaluated for weight (upper panel) and hemorrhagic severity (lower panel). Values represent mean (SE) of six mice from each strain. Asterisk indicates significant difference from corresponding saline-treated strain, p < .05, according to Dunnett's multiple comparison test.
effects of CdCI2, as reported previously (Taylor et al., 1973; Nolan et al., 1986). Strain Comparison of CdMT Toxicity Tissue Concentrations of Cd Accumulation of Cd in liver and kidney following CdMT administration in the four mice strains is depicted in Fig. 3. Kidneys of all strains sequestered the majority of Cd, administered as CdMT. There were no significant differences in Cd renal content between any of the strains, with the exception of the C57 substrain at a dose level of 0.4 mg/kg. Hepatic tissue accumulated much less Cd than did renal tissue. There were no significant differences between strains in Cd content of the liver at any dose level tested.
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Cd was not measurable in testicular tissues following CdMT treatment in all strains at all dose levels (data not shown). Tissue Levels of Metallothionein The ability of hepatic and renal tissues to synthesize MT in response to CdMT is depicted in Fig. 4. The four strains of mice did not differ markedly in their capacity to synthesize MT in hepatic tissue over the dose range of 0.2 to 1.6 mg/kg. The ability of renal tissues to synthesize MT was not significantly different at the three highest dose levels of CdMT, that is, 0.4, 0.8, and 1.6 mg/kg. Only at the lowest dose of 0.2 mg/kg was there any strain difference in the concentration of MT, with the C57 having the highest amount of MT. Renal Toxicity Figure 5 compares the nephrotoxic properties of CdMT among the four substrains. Urinary glucose measurements of
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FIGURE 3. Hepatic and renal tissue Cd concentrations. Mice were treated with CdMT (sc) and tissues collected 24 h later for measurement of hepatic (upper panel) and renal (lower panel) Cd content. Values represent mean (SE) of four to six mice of each strain. Bars with differing letters differ significantly, p < .05, according to Duncan's multiple range test.
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CdMT (mg Cd/kg) FIGURE 4. Hepatic and renal metallothionein (MT) content. Mice were treated with CdMT (sc) and tissues were collected 24 h later for determination of hepatic (upper panel) and renal (lower panel) MT concentrations. Values represent mean (SE) of four to six mice of each strain. Bars with differing letters differ significantly, p < .05, according to Duncan's multiple range test.
C3H, CBA, C57, and DBA strains (top panel) were all similar; the C57 substrain tended to excrete less glucose into the urine after the two highest dosages of CdMT than the other strains, but this difference was not statistically significant. The bottom panel represents the microscopic assessment of renal tissue in the four strains. No discernible differences in the extent of acute proximal tubular necrosis induced by CdMT was observed between any of the murine strains. DISCUSSION Cd accumulates in the liver following acute exposure, inducing metallothionein (MT) synthesis. MT sequesters Cd in the form of a Cdmetallothionein complex, which appears to be released over time into
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the bloodstream, depositing in the proximal tubule cells of the kidney (Dudley et al., 1985). Lysosomal degradation of CdMT in the kidney is suspected to release Cd ions causing cellular damage (Squibb et al., 1984; Webb, 1979). Differences in sensitivities to Cd-induced testicular toxicity among inbred strains of mice led Taylor et al. (1973) to propose that a specific gene governs this response (cadmium resistance, designated cdm). The cdm gene is an autosomal, recessive allele localized on chromosome number 3 of the mouse genome (Taylor et al., 1973). Subsequent investigations have utilized this genetic defect to study the mechanism of Cdinduced testicular and hepatic damage (Hata et al v 1978; Quaife et al.,
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0.4
0.8
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CdMT (mg Cd/kg) FIGURE 5. Renal toxicity induced by CdMT. Mice were treated with CdMT and hydrated 22 h later at a volume of 50 ml/kg. Urine was collected over a 2-h period and assayed for urinary glucose (top panel). Kidneys were scored for severity of acute proximal tubular cell necrosis (bottom panel). Values represent mean (SE) of four to six mice of each strain. Bars with differing letters differ significantly, p < .05, according to Duncan's multiple range test.
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1984; Chellman et al., 1984,1985; Nolan and Shaikh, 1986). We therefore used murine strains that either lacked, or contained, the cdm locus to determine if genetic variations in renal damage to CdMT occurs, as is evident in testicular and hepatic Cd-induced injury. This is the first study to utilize strain variation to Cd in an effort to study renal damage induced by CdMT. Our first series of experiments was designed to substantiate the strain variation to CdCI2-induced testicular and hepatic tissues. C3H mice are extremely susceptible to Cd-induced hepatic toxicity (Tsunoo et al., 1979; Quaife et al., 1984). Our experiments support these observations, as Cd-induced elevations of AST and ALT in the C3H strain were significantly greater than the other three strains tested (Fig. 1). Although the C3H strain is hypersusceptible to Cd-induced liver damage, it is resistant to Cd-induced testicular damage (Taylor et al., 1973). The testes of both the C3H and C57 strains (carriers of the cdm gene) did not appear to be affected by the administration of 25 ^mol Cd/ kg. The testes of both the DBA and CBA mice, strains lacking the cdm gene, were damaged by Cd (Fig. 2). Mice of the C3H strain, carriers of the cdm gene and resistant to the testicular damage caused by Cd, are highly susceptible to liver damage by the same agent. Likewise, the C57 strain, also carriers of the cdm gene, are not protected from Cd-induced hepatic damage. From these results, it appears that, although the cdm locus may contribute to the resistance of Cd-induced testicular toxicity, it has little contribution to Cd-induced hepatotoxicity. Our next series of experiments was designed to test the contribution of the cdm locus to renal damage due to CdMT treatment, by comparing strains that either lack or contain the cdm gene. We measured renal damage by urinary glucose content, as well as microscopic examination. Urinary glucose values between strains were not statistically different. Urine glucose tended to be lower in the C57 mice compared with the three other strains, but the difference was not statistically significant. Histological assessment of kidney tissues indicated no observable differences between mice of all strains over the dose range examined. From these results, as in the case of hepatic damage by Cd, renal damage by CdMT does not have a cdm component. Table 1 compares the presence of the cdm gene to the sensitivity of the testes, liver, and kidney tissues to Cd damage. In the strains tested (CBA/CA, DBA/2J, C3H/HeJ, and C57BI/10), there is no correlation between the presence of the cdm gene and resistance to CdMT-induced renal toxicity. In addition, although the livers of C3H mice are hypersusceptible to Cd, the kidney is not hypersensitive to CdMT. The mechanism for resistance that the cdm gene confers to testicular tissue in response to Cd is not well understood. Hata et al. (1978) examined the ability of DDY-Saitama, DDY(N), DDN-Saitama, STD-DDY, and
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TABLE 1 Presence of cdm Locus and Tissue Susceptibility toCd Tissue SensitivityStrain
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CBA/CA DBA/2J C3H/HeJ C57/BI/10 Note.-,
cdm locus
Testes
Liver
Kidney
+ +
S S R R
S S H S
S S S S
cdm gene absent; +, cdm gene carrier; S, sensitive; R, resistant; H, hypersusceptible.
SWR mice to synthesize metallothionein following Cd administration. No relationship between the degree of MT induction and testicular necrosis was found in these strains. Chellman et al. (1984, 1985) reported that decreased uptake and altered subcellular binding of Cd to an MT-like protein is responsible for resistance to Cd-induced testicular necrosis in strain A/J (resistant) mice compared with strain 129/J (sensitive) mice. Additional studies support the findings that MT, or an MT-like protein, is responsible for testicular strain resistance (Tsunoo et al., 1979; Hata et al., 1980; Hunt and Mhlanga, 1983; Pilez et al., 1983). In contrast, other authors support the contention that there is no correlation between MT and the cdm gene in protecting the testes from Cd damage, or in causing the liver of C3H mice to be hypersusceptible to Cd (Quaife et al., 1986; Nolan and Shaikh, 1986). In summary, the cdm gene does not influence Cd hepatic toxicity or CdMT renal toxicity. Although strain variations to Cd susceptibility are evident in the CBA, DBA, C3H, and C57 strains of mice, no such genetic variation occurs following CdMT administration. REFERENCES Bergmeyer, H. U., Scheibe, P., and Wahlefeld, A. W. 1978. Optimization of methods for aspartate aminotransferase and alanine aminotransferase. Clin. Chem. 24:58-73. Chellman, G. J., Shaikh, Z. A., and Baggs, R. B. 1984. Decreased uptake of altered subcellular disposition of testicular cadmium as possible mechanism of resistance to cadmium-induced testicular necrosis in inbred mice. Toxicology 30:157-169. Chellman, G. J., Shaikh, Z. A., Baggs, R. B., and Diamond, G. L. 1985. Resistance to cadmiuminduced necrosis in testes of inbred mice: Possible role of metallothionein-like cadmiumbinding protein. Toxicol. Appl. Pharmacol. 79:511-523. Dudley, R. E., Gammal, L. M., and Klaassen, C. D. 1985. Cadmium-induced hepatic and renal injury in chronically exposed rats: Likely role of cadmium-metallothionein in nephrotoxicity. Toxicol. Appl. Pharmacol. 77:414-426. Eaton, D. L., and Toal, B. F. 1982. Evaluation of the Cd/hemoglobin affinity assay for the rapid determination of metallothionein in biological tissues. Toxicol. Appl. Pharmacol. 66:134-142. Goering, P. K., and Klaassen, C. D. 1984. Tolerance to cadmium-induced hepatotoxicity following cadmium pretreatment. Toxicol. Appl. Pharmacol. 74:308-313.
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Gunn, S. A., Gould, T. C., and Anderson, W. A. D. 1965. Strain differences in susceptibility of mice and rats to cadmium-induced testicular damage. J. Reprod. Fertil. 10:273-275. Gunn, S. A., Gould, T. C., and Anderson, W. A. D. 1968. Selectivity of organ response to cadmium injury and various protective measures. J. Pathol. Bacteriol. 96:89-96. Hata, A., Tsunoo, H., Nakajima, H., and Kimura, M. 1978. Strain differences in susceptibility of mice to cadmium-induced metallothionein. Toxicol. Lett. 2:45-49. Hata, A., Tsunoo, H., Nakajima, H., Shintau, K., and Kimura, M. 1980. Acute cadmium intoxication in inbred mice: A study on strain differences. Chem. Biol. Interact. 32:29-39. Hunt, D. M., and Mhlanga, T. 1983. Genetic studies on metallothionein synthesis in the mouse: The induction of metallothionein by cadmium in inbred strains. Biochem. Cenet. 21:609-625. Nolan, C. V., and Shaikh, Z. A. 1986. An evaluation of tissue metallothionein and genetic resistance to cadmium toxicity in mice. Toxicol. Appl. Pharmacol. 85:135-144. Onasaka, S., Tanaka, K., Dai, M., and Okahara, K. 1978. A simplified method for determination of metallothionein in animal tissues. Eisei Kagaku 24:128-131. Pilez, J. E., Anderson, R. D., Berry, W., and Herschman, H. R. 1983. Synthesis and degradation of hepatic metallothionein in mice differing in susceptibility to cadmium mortality. Biochem. Cenet. 21:561-578. Plaa, G. L., and Larson, R. E. 1965. Relative nephrotoxic properties of chlorinated methane, ethane, and ethylene derivatives in mice. Toxicol. Appl. Pharmacol. 7:37-44. Quaife, C., Durnam, D., and Mottet, N. K. 1984. Cadmium hypersusceptibility in the C3H mouse liver cell: Cell specificity and possible role of metallothionein. Toxicol. Appl. Pharmacol. 76:917. Squibb, K. S., Pritchard, J. B., and Fowler, B. A. 1984. Cadmium-metallothionein nephropathy: Relationships between ultrastructure/biochemical alterations and intracellular cadmium binding. J. Pharmacol. Exp. Ther. 229:311-321.
Taylor, B. A., Heiniger, H. J., and Meier, H. 1973. Genetic analysis of resistance to cadmium-induced testicular damage in mice. Proc. Soc. Exp. Biol. Med. 143:629-633. Tsunoo, H., Nakajima, H., Hata, A., and Kimura, M. 1979. Genetic influences on induction of metallothionein and mortality from cadmium intoxication. Toxicol. Lett. 4:253-256. Webb, M. 1979. Toxicology of cadmium-thionein. In The Chemistry, Biochemistry, and Biology of Cadmium, ed. M. Webb, pp. 423-431. Elsevier/North Holland, New York. Received May 9, 1991 Accepted August 23, 1991
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Cd‐metallothionein nephrotoxicity in inbred strains of mice L.E. Sendelbach , W.C. Kershaw , F. Cuppage & C.D. Klaassen To cite this article: L.E. Sendelbach , W.C. Kershaw , F. Cuppage & C.D. Klaassen (1992) Cd‐metallothionein nephrotoxicity in inbred strains of mice, Journal of Toxicology and Environmental Health, 35:2, 115-126, DOI: 10.1080/15287399209531600 To link to this article: http://dx.doi.org/10.1080/15287399209531600
Published online: 19 Oct 2009.
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Article views: 1
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Citing articles: 2 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=uteh20 Download by: [NUS National University of Singapore]
Date: 06 November 2015, At: 03:52
Cd-METALLOTHIONEIN NEPHROTOXICITY IN INBRED STRAINS OF MICE
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L. E. Sendelbach, W. C. Kershaw, F. Cuppage, C. D. Klaassen Environmental Health and Occupational Medicine Center, Department of Pharmacology, Toxicology, and Therapeutics, Department of Pathology and Oncology, University of Kansas Medical Center, Kansas City, Kansas
Genetic differences in the acute hepatic and testicular toxicity of Cd occur among different strains of mice. However, it is not known whether genetic variation to the renal damage caused by Cd-metallothionein (CdMT) exists. Therefore, male mice of the C3H/HeJ,C57/B/10, CBA/CA, and DBA/2] strains, previously shown to differ in hepatic and testicular injury due to Cd, were treated with CdMT at dosages of 0.2, 0.4, 0.8, and 1.6 mg/kg (sc). For all strains of mice, tissue accumulation of Cd occurred predominantly in kidney, which had two to three times as much Cd as liver, while testes had no measurable amounts of Cd. Hepatic and renal metallothionein (MT) concentrations were increased with increasing dosage of CdMT, and no differences between strains were demonstrated. Urinary glucose was increased significantly at the three highest dosages of CdMT, with no differences between strains. At each dose level, light microscopic manifestations of CdMT nephropathy did not differ between strains. In summary, all CdMT-treated strains of mice responded similarly with respect to all measured renal parameters (accumulation of Cd and MT and nephrotoxicity). Unlike the strain differences in hepatic and testicular injury from Cd in these strains of mice, CdMT nephrotoxicity shows no such genetic variation.
INTRODUCTION Metallothionein (MT) is a low-molecular-weight protein capable of binding divalent metals. This metal-binding capacity protects experimental animals against Cd toxicity. Cd binds to hepatic MT, forming a cadmium-metallothionein (CdMT) complex within liver that renders the ion nontoxic (Goering and Klaassen, 1984). Some of the CdMT complex is released from hepatic tissue and eventually deposits within the proximal tubule cells of the kidney (Dudley et al., 1985). Relocation of CdMT from This work was supported by the U.S. Public Health Service grant ES-01142. Presented in part at the 27th Annual Meeting of the Society of Toxicology, February 1988, Dallas, Tex. L. E. Sendelbach was supported by U.S. Public Health Service training grant ES-07079, and has a current address of 57 Union Street, Worcester, MA 01608. W. C. Kershaw was supported by U.S. Public Health Service grant ES-07079, and has a current address of Miami Valley Laboratories, Procter & Gamble Company, Cincinnati, OH 45239. Requests for reprints should be sent to C. D. Klaassen, Ph.D., Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, 39th and Rainbow Blvd., Kansas City, KS 66160-7417. 115 Journal of Toxicology and Environmental Health, 35:115-126, 1992 Copyright © 1992 by Hemisphere Publishing Corporation
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the liver to the kidney might be responsible for the chronic toxicity of Cd seen in humans and experimental animals. In contrast to the nephrotoxic potential of CdMT, the target tissues of inorganic Cd (e.g., CdCl2) are testes and liver. Genetic differences of Cd toxicity in testes and liver have been demonstrated in certain strains of mice. Gunn et al. (1965,1968) have shown the testes of rodents to be particularly sensitive to inorganic Cd. In addition, some strains of mice show particular sensitivities to Cd-induced testicular damage. The AJ, BALB/CJ, C57BL/6J, and C3H/HeJ strains are resistant to testicular damage, whereas the 129/J, AKR/J, CBA/J, and C57BR/cdJ are sensitive (Gunn et al., 1965, 1968; Hata et al. 1978; Chellman et al., 1984, 1985; Nolan and Shaikh, 1986). The differences in sensitivities of murine strains to Cd-induced testicular damage lead Taylor to propose that this response is genetically controlled by a single autosomal recessive gene designated cdm (Taylor et al., 1973). Strain variations in the response of hepatic tissue of inorganic Cd are evident, as well. The C3H strain is particularly sensitive to Cd-induced hepatotoxicity (Quaife et al., 1984), whereas the DBA and BALB/c strains are more resistant to the hepatotoxic action of Cd (Tsunoo et al., 1979). However, those strains of mice that are resistant to hepatic damage are not necessarily resistant to testicular damage caused by Cd. For example, the C3H strain is resistant to Cd-induced testicular damage but hypersensitive to hepatic damage. Although genetic variation to the hepatic and testicular damage of Cd is evident in certain strains of mice, there is no known genetic variation to the renal damage caused by CdMT. Because CdMT is the organic form of Cd suspected to be responsible for chronic nephrotoxicity (Dudley et al., 1985), a genetic analysis of murine strain variation might be beneficial in understanding the mechanism of chronic Cd disease. Therefore, mice of the C3H/HeJ, C57BL/10, CBA/CA, and DBA/ 2J strains, previously shown to differ in hepatic and testicular injury due to Cd, were treated with CdMT to test for strain variation to this form of Cd.
METHODS Animals Male C3H/HeJ, C57BL/10, CBA/CA, and DBA/2J mice were received from Jackson Laboratories (Bar Harbor, Me.). All animals were 6 wk of age at receipt, and housed for a 2-week acclimation period. Animals were group housed in plastic shoebox cages on hardwood chip bedding. Fluorescent lighting was maintained on a 12-h on/off cycle. Food, in pellet form (Purina Rodent Laboratory Chow 5001, Ralston Purina Co., St. Louis,
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Mo.), and water were provided ad libitum. The temperature of the animal room was maintained at 23 ± 1°C. Mice were injected with CdCI2 (25 /*mol/kg, sc, n = 6), 109CdMT (0.2, 0.4, 0.8, and 1.6 mg Cd/kg, sc, n = 6), or isotonic saline as vehicle controls (n = 4). Serum was collected for evaluation of transaminase activity 24 h following CdCI2 or CdMT administration. Samples were stored at 4°C and assayed within 24 h of collection. Immediately following serum collection, testes and livers were then removed and weighed. Kidneys were then removed and stored in 10% neutral buffered formalin. Kidneys were routinely processed, stained with hematoxylin and eosin, and evaluated microscopically. Chemicals Cadmium chloride was obtained from Fisher Scientific Co. (St. Louis, Mo.). Carrier-free 109Cd was obtained from New England Nuclear (Boston, Mass.). The reagents used for the determination of glucose and creatinine in urine as well as the activities of alanine (ALT) and aspartate aminotransferases (AST) in serum were supplied as kits (Sigma Chemical Co., St. Louis, Mo.). All other chemicals were of reagent grade. Preparation of Cd-Metallothionein Male rats received 3 mg Cd/kg, sc, plus 1.6 /xCi 109Cd/kg once a day, 3 times per week for 3 wk. Rats were decapitated and the livers were excised and homogenized in a Potter-Elvehjem glass homogenizer, using a Teflon pestle, in a 1 :1 volume of Tris-acetate buffer (pH 7.4, 4°C). The homogenate was then centrifuged at 10,000 x g for 10 min (4°C) in a Sorvall RC2-B centrifuge (Sorvall Instruments, Norwalk, Conn.). The supernatant was heat treated at 80°C for 5 min followed by centrifugation at 15,000 x g for 20 min. This supernatant was then applied to a Sephadex G-75 column (2.6 x 60 cm) and eluted with 10 mM "iris-acetate buffer (pH 7.4, 4°C). The MT peak obtained from the column, with a relative elution volume (Ve/Vo) of approximately 2, was collected, applied to a DEAE Sephadex A-25 column (2.6 x 35 cm), and eluted with a linear gradient of 10-240 mM Tris-acetate buffer (pH 7.4, 4°C). Fractions from the second peak (MT-II) were pooled, desalted through G-25 columns, and lyophilized. Tissue Cd Concentration Portions of the testes, kidney, and liver were assayed for 109Cd radioactivity by means of a Packard model 5130 Autogamma scintillation counter (Packard Instrument Co., Downers Grove, III.), and tissue Cd contents were calculated using the specific activity of the 109Cd-MT dosing solutions.
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Metallothionein Analysis Mice were necropsied 24 h following CdMT administration and the testes, kidney, and liver were excised for determination of MT content. Metallothionein determinations were performed by the Cd-hemoglobin assay of Onasaka et al. (1978) as described by Eaton and Toal (1982).
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Urinalysis Mice were administered Cd-MT, and hydrated 22 h later at a volume of 50 ml/kg. Urine was collected over a 2-h period according to the method of Plaa and Larson (1965) without filter paper. Biochemical Assays Serum activities of ALT and AST were determined according to the method of Bergmeyer et al. (1978). Urinary glucose (Sigma technical bulletin 15-UV, 1983) and creatinine (Sigma procedure 555) were determined using commercially available kits (Sigma Chemical Co.). Macro and Microscopic Assessment of Testicular and Renal Tissues Testes were evaluated for surface hemorrhage at gross necropsy. Grade scores of 1, 2, 3, 4, and 5 corresponded to 0-5, 5-25, 25-50, 50-75, and >75%, respectively, of the testicular surface being hemorrhagic. Kidneys were fixed in 10% neutral buffered formalin, routinely processed, sectioned at 6 /*m, and stained with hematoxylin and eosin for microscopic assessment. Lesions were scored, using 1 to indicate normal, 2 for focal renal tubular necrosis (75%) of the proximal tubule with all components of the tubules necrotic with multiple casts in distal segments of the nephrons. Statistics Data are presented as mean ± (SE). The data were initially compared using a one-way analysis of variance. When significant differences were found, intrastrain comparisons were made using the post hoc procedure of Dunnett's multiple comparison test; interstrain comparisons were performed by the post hoc procedure of Duncan's multiple range test. Differences were considered significant at p < .05. RESULTS Strain Comparison of CdCI2 Toxicity Hepatotoxicity Figure 1 illustrates the hepatic toxicity of CdCI2 in C3H, CBA, DBA, and C57 strains of mice, as measured by the serum activities of ALT and AST. CdCI2, at 25 jumol/kg, increased the activities of
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Saline
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CdCl,
FIGURE 1. Hepatic injury produced by CdCI2. Mice were treated with CdCI2 (25 ftmol/kg, sc), and sera were collected 24 h later for measurement of aspartate aminotransferase (AST, upper panel), and alanine aminotransferase (AST, lower panel) activities. Values represent means (SE) of six mice from each strain. Bars with differing letters differ significantly, p < .05, according to Duncan's multiple range test.
both serum enzymes in all four strains of mice 24 h following administration. CdCI2 produced a greater increase in ALT and AST in the C3H strain than in the other strains. Thus the C3H strain is hypersusceptible to the hepatic effects of Cd, as had been demonstrated previously (Quaife et al., 1984). Testicular Toxicity Strain variation to testicular toxicity of CdCI2 is depicted in Figure 2. The toxicity of CdCI2 to testicular tissues was determined quantitatively by an increase in testicular weights and qualitatively by severity of testicular surface hemorrhage. Both measurements of toxicity were increased in the CBA and DBA mice, strains known to be sensitive to the testicular effects of Cd (Taylor et al., 1973; Nolan et al., 1986). The C3H and C57 murine strains were resistant to the testicular
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300
Saline
CdCU
FIGURE 2. Testicular damage induced by CdCI2. Mice were treated with CdCI2 (25 ^mol/kg, sc), and testes were collected 24 h later. Testes were evaluated for weight (upper panel) and hemorrhagic severity (lower panel). Values represent mean (SE) of six mice from each strain. Asterisk indicates significant difference from corresponding saline-treated strain, p < .05, according to Dunnett's multiple comparison test.
effects of CdCI2, as reported previously (Taylor et al., 1973; Nolan et al., 1986). Strain Comparison of CdMT Toxicity Tissue Concentrations of Cd Accumulation of Cd in liver and kidney following CdMT administration in the four mice strains is depicted in Fig. 3. Kidneys of all strains sequestered the majority of Cd, administered as CdMT. There were no significant differences in Cd renal content between any of the strains, with the exception of the C57 substrain at a dose level of 0.4 mg/kg. Hepatic tissue accumulated much less Cd than did renal tissue. There were no significant differences between strains in Cd content of the liver at any dose level tested.
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Cd was not measurable in testicular tissues following CdMT treatment in all strains at all dose levels (data not shown). Tissue Levels of Metallothionein The ability of hepatic and renal tissues to synthesize MT in response to CdMT is depicted in Fig. 4. The four strains of mice did not differ markedly in their capacity to synthesize MT in hepatic tissue over the dose range of 0.2 to 1.6 mg/kg. The ability of renal tissues to synthesize MT was not significantly different at the three highest dose levels of CdMT, that is, 0.4, 0.8, and 1.6 mg/kg. Only at the lowest dose of 0.2 mg/kg was there any strain difference in the concentration of MT, with the C57 having the highest amount of MT. Renal Toxicity Figure 5 compares the nephrotoxic properties of CdMT among the four substrains. Urinary glucose measurements of
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FIGURE 3. Hepatic and renal tissue Cd concentrations. Mice were treated with CdMT (sc) and tissues collected 24 h later for measurement of hepatic (upper panel) and renal (lower panel) Cd content. Values represent mean (SE) of four to six mice of each strain. Bars with differing letters differ significantly, p < .05, according to Duncan's multiple range test.
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CdMT (mg Cd/kg) FIGURE 4. Hepatic and renal metallothionein (MT) content. Mice were treated with CdMT (sc) and tissues were collected 24 h later for determination of hepatic (upper panel) and renal (lower panel) MT concentrations. Values represent mean (SE) of four to six mice of each strain. Bars with differing letters differ significantly, p < .05, according to Duncan's multiple range test.
C3H, CBA, C57, and DBA strains (top panel) were all similar; the C57 substrain tended to excrete less glucose into the urine after the two highest dosages of CdMT than the other strains, but this difference was not statistically significant. The bottom panel represents the microscopic assessment of renal tissue in the four strains. No discernible differences in the extent of acute proximal tubular necrosis induced by CdMT was observed between any of the murine strains. DISCUSSION Cd accumulates in the liver following acute exposure, inducing metallothionein (MT) synthesis. MT sequesters Cd in the form of a Cdmetallothionein complex, which appears to be released over time into
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the bloodstream, depositing in the proximal tubule cells of the kidney (Dudley et al., 1985). Lysosomal degradation of CdMT in the kidney is suspected to release Cd ions causing cellular damage (Squibb et al., 1984; Webb, 1979). Differences in sensitivities to Cd-induced testicular toxicity among inbred strains of mice led Taylor et al. (1973) to propose that a specific gene governs this response (cadmium resistance, designated cdm). The cdm gene is an autosomal, recessive allele localized on chromosome number 3 of the mouse genome (Taylor et al., 1973). Subsequent investigations have utilized this genetic defect to study the mechanism of Cdinduced testicular and hepatic damage (Hata et al v 1978; Quaife et al.,
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0.4
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1.6
CdMT (mg Cd/kg) FIGURE 5. Renal toxicity induced by CdMT. Mice were treated with CdMT and hydrated 22 h later at a volume of 50 ml/kg. Urine was collected over a 2-h period and assayed for urinary glucose (top panel). Kidneys were scored for severity of acute proximal tubular cell necrosis (bottom panel). Values represent mean (SE) of four to six mice of each strain. Bars with differing letters differ significantly, p < .05, according to Duncan's multiple range test.
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1984; Chellman et al., 1984,1985; Nolan and Shaikh, 1986). We therefore used murine strains that either lacked, or contained, the cdm locus to determine if genetic variations in renal damage to CdMT occurs, as is evident in testicular and hepatic Cd-induced injury. This is the first study to utilize strain variation to Cd in an effort to study renal damage induced by CdMT. Our first series of experiments was designed to substantiate the strain variation to CdCI2-induced testicular and hepatic tissues. C3H mice are extremely susceptible to Cd-induced hepatic toxicity (Tsunoo et al., 1979; Quaife et al., 1984). Our experiments support these observations, as Cd-induced elevations of AST and ALT in the C3H strain were significantly greater than the other three strains tested (Fig. 1). Although the C3H strain is hypersusceptible to Cd-induced liver damage, it is resistant to Cd-induced testicular damage (Taylor et al., 1973). The testes of both the C3H and C57 strains (carriers of the cdm gene) did not appear to be affected by the administration of 25 ^mol Cd/ kg. The testes of both the DBA and CBA mice, strains lacking the cdm gene, were damaged by Cd (Fig. 2). Mice of the C3H strain, carriers of the cdm gene and resistant to the testicular damage caused by Cd, are highly susceptible to liver damage by the same agent. Likewise, the C57 strain, also carriers of the cdm gene, are not protected from Cd-induced hepatic damage. From these results, it appears that, although the cdm locus may contribute to the resistance of Cd-induced testicular toxicity, it has little contribution to Cd-induced hepatotoxicity. Our next series of experiments was designed to test the contribution of the cdm locus to renal damage due to CdMT treatment, by comparing strains that either lack or contain the cdm gene. We measured renal damage by urinary glucose content, as well as microscopic examination. Urinary glucose values between strains were not statistically different. Urine glucose tended to be lower in the C57 mice compared with the three other strains, but the difference was not statistically significant. Histological assessment of kidney tissues indicated no observable differences between mice of all strains over the dose range examined. From these results, as in the case of hepatic damage by Cd, renal damage by CdMT does not have a cdm component. Table 1 compares the presence of the cdm gene to the sensitivity of the testes, liver, and kidney tissues to Cd damage. In the strains tested (CBA/CA, DBA/2J, C3H/HeJ, and C57BI/10), there is no correlation between the presence of the cdm gene and resistance to CdMT-induced renal toxicity. In addition, although the livers of C3H mice are hypersusceptible to Cd, the kidney is not hypersensitive to CdMT. The mechanism for resistance that the cdm gene confers to testicular tissue in response to Cd is not well understood. Hata et al. (1978) examined the ability of DDY-Saitama, DDY(N), DDN-Saitama, STD-DDY, and
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TABLE 1 Presence of cdm Locus and Tissue Susceptibility toCd Tissue SensitivityStrain
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CBA/CA DBA/2J C3H/HeJ C57/BI/10 Note.-,
cdm locus
Testes
Liver
Kidney
+ +
S S R R
S S H S
S S S S
cdm gene absent; +, cdm gene carrier; S, sensitive; R, resistant; H, hypersusceptible.
SWR mice to synthesize metallothionein following Cd administration. No relationship between the degree of MT induction and testicular necrosis was found in these strains. Chellman et al. (1984, 1985) reported that decreased uptake and altered subcellular binding of Cd to an MT-like protein is responsible for resistance to Cd-induced testicular necrosis in strain A/J (resistant) mice compared with strain 129/J (sensitive) mice. Additional studies support the findings that MT, or an MT-like protein, is responsible for testicular strain resistance (Tsunoo et al., 1979; Hata et al., 1980; Hunt and Mhlanga, 1983; Pilez et al., 1983). In contrast, other authors support the contention that there is no correlation between MT and the cdm gene in protecting the testes from Cd damage, or in causing the liver of C3H mice to be hypersusceptible to Cd (Quaife et al., 1986; Nolan and Shaikh, 1986). In summary, the cdm gene does not influence Cd hepatic toxicity or CdMT renal toxicity. Although strain variations to Cd susceptibility are evident in the CBA, DBA, C3H, and C57 strains of mice, no such genetic variation occurs following CdMT administration. REFERENCES Bergmeyer, H. U., Scheibe, P., and Wahlefeld, A. W. 1978. Optimization of methods for aspartate aminotransferase and alanine aminotransferase. Clin. Chem. 24:58-73. Chellman, G. J., Shaikh, Z. A., and Baggs, R. B. 1984. Decreased uptake of altered subcellular disposition of testicular cadmium as possible mechanism of resistance to cadmium-induced testicular necrosis in inbred mice. Toxicology 30:157-169. Chellman, G. J., Shaikh, Z. A., Baggs, R. B., and Diamond, G. L. 1985. Resistance to cadmiuminduced necrosis in testes of inbred mice: Possible role of metallothionein-like cadmiumbinding protein. Toxicol. Appl. Pharmacol. 79:511-523. Dudley, R. E., Gammal, L. M., and Klaassen, C. D. 1985. Cadmium-induced hepatic and renal injury in chronically exposed rats: Likely role of cadmium-metallothionein in nephrotoxicity. Toxicol. Appl. Pharmacol. 77:414-426. Eaton, D. L., and Toal, B. F. 1982. Evaluation of the Cd/hemoglobin affinity assay for the rapid determination of metallothionein in biological tissues. Toxicol. Appl. Pharmacol. 66:134-142. Goering, P. K., and Klaassen, C. D. 1984. Tolerance to cadmium-induced hepatotoxicity following cadmium pretreatment. Toxicol. Appl. Pharmacol. 74:308-313.
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Gunn, S. A., Gould, T. C., and Anderson, W. A. D. 1965. Strain differences in susceptibility of mice and rats to cadmium-induced testicular damage. J. Reprod. Fertil. 10:273-275. Gunn, S. A., Gould, T. C., and Anderson, W. A. D. 1968. Selectivity of organ response to cadmium injury and various protective measures. J. Pathol. Bacteriol. 96:89-96. Hata, A., Tsunoo, H., Nakajima, H., and Kimura, M. 1978. Strain differences in susceptibility of mice to cadmium-induced metallothionein. Toxicol. Lett. 2:45-49. Hata, A., Tsunoo, H., Nakajima, H., Shintau, K., and Kimura, M. 1980. Acute cadmium intoxication in inbred mice: A study on strain differences. Chem. Biol. Interact. 32:29-39. Hunt, D. M., and Mhlanga, T. 1983. Genetic studies on metallothionein synthesis in the mouse: The induction of metallothionein by cadmium in inbred strains. Biochem. Cenet. 21:609-625. Nolan, C. V., and Shaikh, Z. A. 1986. An evaluation of tissue metallothionein and genetic resistance to cadmium toxicity in mice. Toxicol. Appl. Pharmacol. 85:135-144. Onasaka, S., Tanaka, K., Dai, M., and Okahara, K. 1978. A simplified method for determination of metallothionein in animal tissues. Eisei Kagaku 24:128-131. Pilez, J. E., Anderson, R. D., Berry, W., and Herschman, H. R. 1983. Synthesis and degradation of hepatic metallothionein in mice differing in susceptibility to cadmium mortality. Biochem. Cenet. 21:561-578. Plaa, G. L., and Larson, R. E. 1965. Relative nephrotoxic properties of chlorinated methane, ethane, and ethylene derivatives in mice. Toxicol. Appl. Pharmacol. 7:37-44. Quaife, C., Durnam, D., and Mottet, N. K. 1984. Cadmium hypersusceptibility in the C3H mouse liver cell: Cell specificity and possible role of metallothionein. Toxicol. Appl. Pharmacol. 76:917. Squibb, K. S., Pritchard, J. B., and Fowler, B. A. 1984. Cadmium-metallothionein nephropathy: Relationships between ultrastructure/biochemical alterations and intracellular cadmium binding. J. Pharmacol. Exp. Ther. 229:311-321.
Taylor, B. A., Heiniger, H. J., and Meier, H. 1973. Genetic analysis of resistance to cadmium-induced testicular damage in mice. Proc. Soc. Exp. Biol. Med. 143:629-633. Tsunoo, H., Nakajima, H., Hata, A., and Kimura, M. 1979. Genetic influences on induction of metallothionein and mortality from cadmium intoxication. Toxicol. Lett. 4:253-256. Webb, M. 1979. Toxicology of cadmium-thionein. In The Chemistry, Biochemistry, and Biology of Cadmium, ed. M. Webb, pp. 423-431. Elsevier/North Holland, New York. Received May 9, 1991 Accepted August 23, 1991
